Prophylactic Zinc Administration Combined with Swimming Exercise Prevents Cognitive-Emotional Disturbances and Tissue Injury following a Transient Hypoxic-Ischemic Insult in the Rat

Exercise performance and zinc administration individually yield a protective effect on various neurodegenerative models, including ischemic brain injury. Therefore, this work was aimed at evaluating the combined effect of subacute prophylactic zinc administration and swimming exercise in a transient cerebral ischemia model. The prophylactic zinc administration (2.5 mg/kg of body weight) was provided every 24 h for four days before a 30 min common carotid artery occlusion (CCAO), and 24 h after reperfusion, the rats were subjected to swimming exercise in the Morris Water Maze (MWM). Learning was evaluated daily for five days, and memory on day 12 postreperfusion; anxiety or depression-like behavior was measured by the elevated plus maze and the motor activity by open-field test. Nitrites, lipid peroxidation, and the activity of superoxide dismutase (SOD) and catalase (CAT) were assessed in the temporoparietal cortex and hippocampus. The three nitric oxide (NO) synthase isoforms, chemokines, and their receptor levels were measured by ELISA. Nissl staining evaluated hippocampus cytoarchitecture and Iba-1 immunohistochemistry activated the microglia. Swimming exercise alone could not prevent ischemic damage but, combined with prophylactic zinc administration, reversed the cognitive deficit, decreased NOS and chemokine levels, prevented tissue damage, and increased Iba-1 (+) cell number. These results suggest that the subacute prophylactic zinc administration combined with swimming exercise, but not the individual treatment, prevents the ischemic damage on day 12 postreperfusion in the transient ischemia model.


Introduction
Cerebrovascular disease (CVD) is caused by a temporary or sustained deprivation of oxygen and nutrients in one or more brain areas, leading to cerebral parenchyma injury [1]. This disease affects 15 million people worldwide every year and is one of the leading causes of permanent disability and mortality [2]. In addition, ischemia is a more frequent cause than the hemorrhagic process in CVD [3]. The ischemic process disrupts the blood flow, alters cell homeostasis, and induces immediate cell death by necrosis in the infarct core [4]. Furthermore, in the penumbra zone, the ischemic process triggers deleterious events such as excitotoxicity (release of glutamate and calcium), nitrosative and oxidative stress [5], lytic enzyme activation (proteases, lipases, and nucleases), dynamic cytoskeleton alteration [6], and neuroinflammation [7].
Nevertheless, the constant release of proinflammatory cytokines, chemokines, and adhesion molecules can elicit apoptosis and affect neuroplasticity, which causes cognitive deficits such as learning and memory loss and emotional disturbances such as anxiety or depression-like behavior [32]. Interestingly, some reports have shown that cognitive activity or motor performance protects against neurodegenerative diseases, improving cerebral functionality [33,34]. Swimming exercise, for example, has shown neuroprotection in animal models of Alzheimer [35], spinal muscular atrophy [36], depression [37], and amyotrophic lateral sclerosis, thorough increasing the production and release of growth factors [38]. However, the beneficial effect of exercise performance depends on factors like intensity and frequency to trigger efficient cellular adaptation mechanisms to oxidative stress and increase the antioxidative response stimulated by mitochondrial biogenesis [39,40].
The main beneficial effect of zinc is to promote antioxidant and anti-inflammatory actions against ischemic damage. It modulates the formation of ROS by inhibiting NMDAR through its binding to the GluN2A subunit [53,54]; decreases the reactivity of O 2 due to a structural part of the cytosolic superoxide dismutase (SOD1) [55]; regulates the transcription of reduced glutathione (GSH), SOD, reduced glutathione transferase (GST), and heme oxygenase-(HO-) 1 through Nrf2 [56]; induces BDNF expression [57]; converts pro-BDNF to mature BDNF by activation of zinc-dependent MMPs [58]; prevents the formation of the complex IKK through the interaction of Zn 2+ via influx by Zip8 [59], or the A20 protein [60], inhibiting the NF-κB signaling pathway [61] and decreasing neuroinflammation; and increases the density of neuronal precursor cells after a TBI [62], promoting neurogenesis [63,64]. Furthermore, zinc through microgliaexpressed ZEB1 can regulate neuroinflammation after stroke, decreasing neutrophil chemoattraction by CXCL1 [65].
It has been reported that subacute prophylactic zinc administration in the 10 min CCAO model has a neuroprotective effect, reducing nitrosative stress and cell death [11], increasing chemokine and growth factor levels, and improving learning and memory in the Morris Water Maze (MWM) test [41]. On the contrary, zinc deficiency caused anxiety-like behavior in rats submitted to MWM [66]. Besides, chronic prophylactic zinc (0.2 mg/kg by 14 days) combined with therapeutic selenium administration has a neuroprotective and antioxidant effect on hypoxic-ischemic brain events postreperfusion [42].
Based on the neuroprotection of exercise performance and subacute prophylactic zinc administration, this work was aimed at evaluating whether the combined neuroprotection of prophylactic zinc administration (2.5 mg/kg/24 h for four days) and swimming exercise through MWM can be effective  Figure 1).

Common Carotid Artery Obliteration (CCAO).
The animals were maintained in a sterile room, and surgical instruments were sterilized. Posteriorly, animals were anesthetized with a mixture of ketamine (70 mg/kg) and xylazine (6 mg/ kg) at a dose of 200 μL/100 g of body weight, i.p. After an incision of 0.5 cm long midline skin in the neck area, the left common carotid artery was carefully dissected and occluded for 30 min with a clamp (Bulldog Clamps, INS6000119; Kent Scientific Corporation; Torrington, CT, USA). Upon completion of the occlusion time, the artery reperfusion was visually verified, and the incision was sutured with a 3-0 silk thread (Atramat; Ciudad de Mexico, Mexico). The animals were kept in an individual cage until their complete recovery in the surgery room and then transferred to a temporary vivarium. The animals after experimental processes were euthanized on day 12 postreperfusion using sodium pentobarbital at a dose of 60 mg/kg of body weight [41,42].

Morris Water Maze (MWM)
. MWM pool was used to submit the rats to physical exercise as reported previously [67,68]. Additionally, this test was utilized to evaluate learning and memory. All experimental groups were trained at 24 h after CCAO with MWM to evaluate the learning and memory. The swimming pool was divided into four quadrants, North (N), West (W), South (S), and East (E), and two clues were placed on the inner walls of the tank, which serve as the orientation and location inside the platform. The MWM consisted of a circular tank (swimming pool) of 150 cm diameter and 80 cm height; the escape platform measuring 10 cm in diameter was placed in the quadrant southeast. The tank will be filled with water (19-22°C) one centimeter above the platform height to ensure that rats cannot see the escape platform.
The learning test consisted of four probes per day, one for each quadrant, giving 60 seconds (s) to find the platform (escape latency). Once the rats completed the trial, they remained on the platform for 30 s. Then, they were removed from the platform and waited 30 min to start the subsequent trial. The order of performance of the quadrants was N, W, S, and E, for five consecutive days.
The memory test was evaluated seven days after the last day of the learning test. The memory test was carried out for 60 s in the N quadrant, counting the number of times that the rat passed by the escape platform and the time in which it was localized [42].
2.6. Elevated Plus Maze. The elevated plus maze was used to assess the depressive-like behavior and anxiety of the rats. The maze was placed 90 cm from the ground and consisted of two open arms (25 × 5 × 1 cm; white color) and two closed arms (25 × 5 × 16 cm; black color) connected to a central platform (5 × 5 cm). The open arms had a wall of 1 cm to reduce the risk of falls, whereas the closed arms had a wall of 16 cm to cover the entire arm.
The test was carried out for 5 min, and the number of times that the animal entered the open and closed arms and the time to remain on them were recorded. In addition, the urination and defecation numbers were also quantified.

Open
Field. The open-field test was also used to assess the unrestricted mobility of rats. The evaluations were carried out in a square wood box of 60 cm divided into nine quadrants per side and 70 cm high. The number of times the subject passed through the central quadrant (quadrant 5) and the distance traveled (total number of quadrants visited multiplied by 20 cm) during 5 min were registered.
2.8. Nitric Oxide Quantification. The NO production was assessed by the accumulation of nitrites (NO 2 -) by the modified technique of Griess as described previously [69]. The cerebral cortex and hippocampus were obtained 1 h after the memory test (n = 5 rats in each group), mechanically homogenized in phosphate-buffered saline solution pH 7.4 (PBS), and centrifuged at 12,500 rpm for 30 min at 4°C by using a Z216MK microcentrifuge (HERMLE Labortechnik; Wehingen, Germany). Briefly, the NO concentration was measured in 10 μL of supernatant after the addition of 3 Behavioural Neurology 10 μL of Griess reagent (composed of equal volumes of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride and 1.32% sulfanilamide in 60% acetic acid). In addition, the optical density in a 2 μL sample was measured with a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Bancroft Building, Wilmington, USA) at 540 nm, and the optical density values were interpolated from the standard curve of NaNO 2 (1 to 10 μM) to obtain NO concentration.
2.9. Lipid Peroxidation Quantification. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) as an index of lipid peroxidation were measured in each group (n = 5), following the procedure described previously [69]. The colorimetric reaction was performed in 200 μL of the supernatant supplemented with 650 μL of 10.3 mM N-methyl-2phenyl-indole (Sigma-Aldrich; Saint Louis, MO, USA), which was previously diluted in a mixture of acetonitrile: methanol (3 : 1) and 150 μL of methanesulfonic acid (Sigma-Aldrich; Saint Louis, MO, USA). This reaction mixture was vortexed and incubated at 45°C for 1 h and then centrifuged at 3000 rpm for 10 min. The absorbance of the reaction in supernatant was read at 586 nm m with a SmartSpec 3000 spectrophotometer (Bio-Rad; Hercules, CA, USA). The absorbance values were interpolated from a standard curve of 1,1,3,3tetramethoxypropane (10 mM stock) in the concentration range of 0.25 to 5 μM to calculate the MDA+4-HDA level in the samples.   The tetrazolium salt assays measure mitochondrial activity associated with the electron transport chain, given the rapidity with which most dehydrogenase systems reduce it. Upon reduction, tetrazolium precipitates in the form of a deep red water-insoluble complex. The experimental animals were anesthetized and perfused intracardially with 0.9% isotonic saline solution (SSI); the brains were extracted and coronally sectioned at intervals of 2 mm; then, the sections were incubated for 15 minutes at 37°C in 1% of TTC solution; posteriorly, the tissues were placed overnight in 4% paraformaldehyde solution. After staining, photographs of the brain sections were taken.

Nissl
Staining. Brain slices of 40 μm thick were placed on gelatinized slides, washed with distilled water, and stained with a 0.1% cresyl violet solution (Merck KGaA; San Luis, Missouri, USA) in a 0.25% acetic acid solution for 30 min. Subsequently, the sections were dehydrated in increasing concentrations of ethanol and rinsed with xylene (Hycel; CDMX, Mexico), and Entellan resin (Merck KGaA; Darmstadt, Germany) was used as a mounting medium [11].

Hierarchization Score of the Protective Effect of Pharmacological Strategies (HSPEPS)
. The values of cognitive tests, histopathology, and biochemical probes were expressed according to the modified HSPEPS [69], indicating the efficacy of one or more pharmacological strategies (Register # MX2020010357) treatments. A value lower than healthy was zero, equal to healthy was 1, and higher than healthy was 1.5 or 2, depending on the increasing grade (Table 1). HSPEPS was constructed with the sum of all performance scores of each treatment and study (Table 2).
The HSPEPS analysis on the cognitive tests showed that the CCAO+MWM group had a lesser value (0.5) than the MWM group (4), and the Zn+CCAO+MWM group yielded values closer to basal values (3.5) of the MWM group (Table 3).
In the temporoparietal cortex, nitrite levels were not different among all experimental groups on day 12 postreperfusion (Figure 4(a)). Meanwhile, in the hippocampus, only the zinc group increased the nitrite levels, while the others groups decreased compared with the healthy group (Figure 4(b)).
The SOD activity in the temporoparietal cortex (Figure 4 (e)) and hippocampus (Figure 4(f)) did not show a statistical difference among all groups on day 12 postreperfusion. However, in this region, CCAO injury increased CAT activity (242:02 ± 15:77%, ΦΦ P = 0:0064) compared with the healthy and MWM groups, and the subacute prophylactic zinc administration reverted this increase (77:28 ± 6:58%, † † P = 0:0036) (Figure 4(g)). In the hippocampus, the CAT activity was not statistically different among all experimental groups (Figure 4(h)). ELISA measurements in the temporoparietal cortex showed significantly increased NOS levels compared with Preliminary results showed that the prophylactic zinc administration in the absence of MWM (Zn+CCAO group) caused greater damage revealed by TTC staining at 24 h and 7 days postreperfusion (data not shown). In contrast, the TTC staining in fresh brains on day 12 postreperfusion showed that the evident infarct core in the CCAO hippocampus was prevented by the prophylactic zinc administration combined with swimming (Zn+CCAO+MWM group) ( Figure 6).
Confirming the tissue injury shown by TTC staining (Figure 6), Nissl staining brought about decreased optical density in the three hippocampal layers on day 12 after 30 min CCAO compared with the healthy and MWM groups (Figure 7). Meanwhile, the optical density in the three layers was normalized in the Zn+CCAO+MWM group (Figure 7). In addition, healthy rats submitted to the MWM showed a significant increase in optical density in the hippocampus CA3 layer (Figure 7).
Iba-1 immunohistochemistry was used to explore the response of activated microglia cells in the hippocampus. An increased number of Iba-1-positive cells was consistently found in the three hippocampal layers of the CCAO group compared with the healthy group ( Figure 8). Surprisingly,     The HSPEPS analysis of the histopathological study showed a high score (7.5) in the MWM group compared to the healthy group (5), and this score was reduced to 5 by the subacute prophylactic zinc administration (Zn+MWM group). On the other hand, the lesser score (4) was seen in 10 Behavioural Neurology the CCAO+MWM group, revealing the ischemic damage in the hippocampal CA3 layer. Compared with the former group, the score in the Zn+CCAO+MWM group was higher (7), reaching the score in the MWM group (Table 4).
ELISA assays showed variable chemokine levels in all experimental groups of the temporoparietal cortex ( Figure 9) and hippocampus ( Figure 10). Of relevance to the effect of the combined treatment on ischemia-induced
HSPEPS in biochemical studies included nitrosative stress, NOS isoforms, antioxidant activity, and chemokines. This analysis showed that swimming (MWM group) yielded a higher score (17) than the healthy group (13) that was unmodified by the subacute prophylactic zinc administration (Zn+MWM) and the cerebral ischemia (CCAO+MWM) groups. However, the subacute prophylactic zinc administration combined with a 5-day swimming exercise produced a score (13) similar to the healthy value (Table 5).

Discussion
This work focused on a transient moderate cerebral ischemia whose functional deficiency could still be at least partially reversed by treatments. In particular, the work explored the effect of subacute prophylactic zinc administration combined with swimming exercise on biochemical and cellular alterations in the late stage of ischemia. In this stage, the neurological deficit persists regardless of neuroinflammation which was naturally resolved by the end of the early stage (before two weeks postreperfusion). Our results prove that subacute prophylactic zinc administration combined with a 5-day swimming exercise in MWM avoided the cognitive and emotional deficits commonly presented in the clinic [70,71], but not motor activity. Furthermore, preventing those deficits is consistent with reducing tissue injury and promoting neuroregeneration in the hippocampus, as TTC and Nissl staining showed. On this basis, it is thought that the increased Iba-1 cell (+) density in the hippocampus would reflect the participation of M2 phenotype microglia trying to remove cell detritus rather than the neuroinflammatory process endurance. Similarly, the increase in nitrosative stress, nNOS, iNOS, and CAT activity in the temporoparietal cortex in the CCAO group might reflect a late postischemic sequel in this region that can account for the reduced free mobility in the open field and its resistance to treatment with the subacute prophylactic zinc administration combined with swimming at 30 days postreperfusion. Preliminary studies show that 2.5 mg/kg subacute prophylactic administration in the absence of swimming caused severe cerebral damage after CCAO at 24 h and 7 days postreperfusion (data not shown). This harmful effect can be caused by the higher zinc concentration accumulated in the brain [49] due to the zinc treatment and the released zinc from presynaptic vesicles following long times of artery occlusion, thus leading to excitotoxicity [72]. On this basis, we utilized the zinc treatment combined with swimming exercise in a transient ischemic process of 30 min.

Swimming Exercise and Nitrosative
Stress. Different modalities of swimming exercise are known to elicit a preconditioning effect [73,74], able to attenuate or reverse cerebral injury in the early stage after ischemia [75], and improve cognition [76]. Furthermore, NO production from the three NOS isoforms [77] and increased hydroxyl radical rate [78] triggered by aerobic exercise participate in the formation of associative and spatial memory [79]. Our results in the temporoparietal cortex in the MWM group showed that swimming exercise during the late stage of ischemia increased lipid peroxidation, iNOS, and eNOS, events also associated with improved sensory and motor skills [80], spatial recognition, and long-term learning and memory [81]. Altogether, these findings support that NO plays a critical role in synaptic transmission, acting as a retrograde neurotransmitter [79], and that ROS are required in LTP formation, increasing memory acquisition and consolidation [82,83].
We found that rats with 30 min CCAO submitted to swimming exercise presented learning and memory loss on day 12 postreperfusion, showing that swimming exercise alone was insufficient to avoid cerebral ischemic damage. In contrast, the prevention of cognitive deficits and tissue damage was achieved with the addition of subacute prophylactic zinc administration. eNOS likely mediates this beneficial effect since exercise prevents vascular endothelium dysfunction [77,84,85], reduces infarct volume [86], and promotes nerve repair after cerebral ischemia [77]. Furthermore, zinc stabilizing eNOS increases its enzymatic activity [87]. On the contrary, zinc deficiency increases O 2 levels by eNOS, promoting cerebral damage [88], as could occur in the CCAO group.
An increasing number of reports have associated the protective effect of zinc with swimming exercise stimulation and improved cell functioning [89,90]. Accordingly, swimming training using MWM protects hippocampal neurons against degenerative changes and improves learning and memory [51]. Furthermore, oral supplementation of 16 or 32 ppm ZnSO 4 [91,92] and subacute intraperitoneal administration of 2.5 mg/kg of ZnCl 2 [41] or 0.2 mg/kg chronically administered [42] prevent memory loss in ischemia or TBI [41,42,51]. The protective effect of exercise and zinc is proposed to be mediated by increasing the expression of growth factors [41] through regulating CREB [67] and antioxidant enzymes, such as SOD [42] and CAT, in animal damage models [93,94]. These neuroprotective events are shown to occur in the early stage (day 7) postreperfusion when ROS is elevated by the ischemic process [95,96], as confirmed by unpublished results of our group. However, in the late stage (day 12), zinc and swimming exercise did not modify SOD and CAT activity in the hippocampus but decreased the CCAO-activated CAT activity in the temporoparietal cortex. These results suggest that the increased CAT activity in the ischemic group reflects a response to oxidative stress, which is prevented by zinc combined with swimming. In addition, zinc decreases CAT activity in neonate rats [69] through the regulation of ERK phosphorylation [97]. Furthermore, zinc can exert an anxiolytic effect [98] by decreasing the inflammatory process [99], nNOS [100], and cell damage [99]; increasing antioxidant enzymes [101], BDNF [102], and synaptic plasticity; modulating neurochemical transmission [103]; regulating the activity of NMDA receptors [103][104][105]; and reducing the ischemic damage [106].
In addition, several studies show that the administration of NMDA receptor antagonists prevented tissue damage [107,108] and modified some aspects of behavior [108], which can be enhanced by hypothermia caused by anesthesia [109]. In this work, we used ketamine which has been useful in models of ischemia, being capable of reducing the damage when administered superacute and therapeutically mode [110,111], due to its inhibitor effect on glutamate receptors [112].
Accordingly, subacute prophylactic zinc administration combined with swimming increased the entry to the open arms in the elevated plus maze, thus showing that the combined treatment prevented the CCAO-induced anxiety. However, the combined treatment could not improve motor skills in the open field, as reported in the TBI model [51]. These results show that the subacute prophylactic zinc administration combined with swimming zinc partially protects the sequel of ischemic damage on day 30 after reperfusion.

Zinc Administration Combined with Swimming Prevents
Tissue Injury. The 30 min hypoxia-ischemia model causes neuronal death that remains until seven days in the infarct core [24], whereas in the penumbra zone, highly plastic events triggered lead to tissue regeneration 14 days postreperfusion [27]. Our results agree with both events. First, the CCAO group showed signs of infarct on day 12 postreperfusion, revealed by the decreased cell density mainly in DG and CA3 layers of the hippocampus that coincides with the reduced cell viability. Second, the recovery of cell density and viability in the hippocampus in the Zn+CCAO group indicates that the subacute prophylactic zinc administration induced potentiation of plastic events. A similar effect was observed in a 10 min CCAO model [11], thus showing that the zinc neuroprotector effect combined with swimming is still effective in a cerebral injury of 30 min CCAO, reported previously in a 10 min model [11,41,42].
Tissue recovery from CNS injury also involves the participation of microglia [113]. The CCAO group showed an increased Iba-1(+) cell number to the healthy group on day 12 postreperfusion, suggesting that the natural course of both neuroinflammation and tissue recovery has yet not terminated on day 12 postreperfusion. However, subacute prophylactic zinc administration combined with swimming increased the Iba-1 (+) cell population over that of the CCAO group on day 12 postreperfusion. This result agrees with the previous report showing that the Iba-1 (+) cell number is still high at this time and decreased at 14 days postreperfusion [27]. However, subacute prophylactic zinc administration combined with swimming decreased the protein levels of chemokines (CXCL1, CCL2, CXCL13, and CX3CL1), the three isoforms of NOS, and the tissue damage caused by CCAO, showing a decrease in the neuroinflammatory process. These results also suggest that the combined treatment modulated the inflammation process, decreasing chemoattraction of leukocytes such as neutrophils by CXCL1 [114], macrophage/microglia by CCL2 [115] and CX3CL1 [116], and lymphocyte B, natural killer cells [117], and M1 microglia activation [118] by CXCL13 [119]. Therefore, the increase in microglial cells does not indicate a harmful effect but their participation in removing cell detritus.
In contrast, some reports have shown that these chemokines also have a neuroprotector effect in the late phase postreperfusion. For instance, CCL2 chemoattracts neuroblasts [120] and promotes neurogenesis [121] and synaptic plasticity in the hippocampus [122], thus decreasing the infarct size and recovering the cognitive function [41,123]. In addition, CXCL1 promotes the remyelination process through the migration and maturation of oligodendrocytes [124]. Furthermore, CXCL13 induces proliferation and autophagy of mesenchymal stem cells [125] and has a chemoattractant action of neuroblast [126], and CX3CL1 causes the chemoattraction and differentiation of neural precursor cells [127] and reduces the microglia reactivity, decreasing neuroinflammation [128,129]. However, the decrease in these chemokines by prophylactic zinc administration combined with zinc could affect those recovery processes in the long term.
The HSPEPS analysis reported previously [69] shows that in the CCAO+MWM group, the tissue damage correlates with the cognitive dysfunction, although biochemically; there are no differences with the MWM group. The MWM group showed a more beneficial effect on hippocampal cytoarchitecture and biochemical variables than the healthy control group. However, the subacute zinc prophylactic administration blocked the cognitive deficit and tissue damage caused by CCAO, and such effect was unmodified on the biochemical variables. In contrast, the score equality to the healthy control and MWM values observed in the Zn +CCAO+MWM in all the tests shows that the prophylactic zinc administration combined with swimming exercise prevented the 30 min CCAO-induced ischemic damage. In summary, HSPEPS analysis proves that the individual treatment did not cause the expected effect, i.e., a preconditioning effect by the prophylactic zinc administration or the therapeutic effect by swimming. However, effective neuroprotection against ischemic damage induced by 30 min CCAO in the rat was reached by combining the prophylactic and therapeutic strategies.

Conclusion
Prophylactic zinc administration combined with swimming exercise prevented ischemic damage in the 30 min CCAO model by decreasing neuroinflammation and nitrosative stress, preventing cell death, and improving the spatial learning-memory on day 12 postreperfusion and anxietylike behavior on day 30 postreperfusion. These results suggest that subacute prophylactic zinc administration requires another complementary therapeutic approach to maintain the neuroprotector effect in the late phase of cerebral hypoxia-ischemia. 15 Behavioural Neurology

Data Availability
Data is included in this manuscript.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
Ana Karina Aguilar-Peralta and Alejandro Gonzalez-Vazquez were responsible for the methodology, formal analysis, investigation, writing original draft, review, and editing. Victor M. Blanco-Alvarez was responsible for the validation and investigation. Constantino Tomas-Sanchez was responsible for validation and investigation. Juan A. Gonzalez-Barrios was responsible for resources and funding acquisition. Daniel Martinez-Fong was responsible for animal supply, writing, review, and editing. Guadalupe Soto-Rodriguez was responsible for the methodology. Eduardo Brambila was responsible for formal statistical analysis. Lourdes Millán-Perez Peña was responsible for the resources. Viridiana Vargas-Castro helped in revision of article, Bertha Alicia Leon-Chavez was responsible for conceptualization, project administration, visualization, supervision, funding acquisition, writing, review, and editing.